PROJECT ABSTRACT Molecular and genetic analysis of novel Slicer-dependent miRNA pathways in blood Most conserved microRNAs (miRNAs) are generated by a biogenesis pathway that deposits them into an Argonaute effector, guiding them to broad regulatory target networks. Amongst the cohort of four mammalian Argonautes, only Ago2 has catalytic ability to cleave transcripts, an enzymatic activity known as Slicing that underlies experimental RNA interference. Nevertheless, the endogenous biological usage of mammalian Slicing remains largely mysterious. Our previous and ongoing studies provide the unexpected perspective of multiple Slicing-dependent biogenesis strategies that generate both Dicer-independent and Dicer-dependent erythroid miRNAs. These data strongly support our hypothesis that a dominant usage of Ago2 catalysis is to generate specific conserved miRNAs in the blood system. Our extensive preliminary data are the basis of (1) a series of biochemical and genomic experiments to elucidate a novel Slicing-dependent miRNA biogenesis mechanism, (2) genetic studies of novel knockout animals of erythroid, Slicing-dependent miRNAs in normal development, blood homeostasis and leukemia, and (3) molecular genetic analyses that seek to connect dysregulated processes in Ago2-catalytically defective blood system to specific Slicing- dependent miRNAs. These studies will bring new insights on post-transcriptional control of erythroid development, homeostasis, and blood cancer, as well as pinpoint the functional basis of mammalian RNAi to the generation of erythroid-specific miRNAs.